The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.     

Phospholipid metabolism of stimulated lymphocytes. Comparison of the activation of acyl-CoA:lysolecithin acyltransferase with the binding of concanavalin A to thymocytes.

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA:1-acylglycero-3-phosphorylcholine-O-acyltransferase, EC 2.3.1.23) was determined as also the binding of 125I-labelled concanavalin A to intact cells and isolated membranes. The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 degrees C. The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Correspondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8). These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Inhibition of acyl coenzyme A:lysolecithin acyltransferases by local anesthetics, detergents and inhibitors of cyclic nucleotide phosphodiesterases.

Acyl coenzyme A:lysolecithin acyltransferase plays a major role in regulating the amount of lysolecithin in cell membranes. The acyltransferase activity in microsomal preparations from rat liver, rat heart and rabbit gastric mucosa is inhibited by a series of tertiary amine local anesthetics, detergents, and some inhibitors of cyclic nucleotide phosphodiesterases. Aspirin and indomethacin cause elevated lysolecithin/lecithin ratios in the stomachs of mice after oral administration. Inhibition of acyltransferase activity in microsomal preparations by local anesthetics correlates with reported anesthetic potencies at approximately $\text{1}\text{100}$ reported therapeutic dosages. In BHK-13 cells acyltransferase activity is inhibited at $\text{1}\text{3}$ to $\text{1}\text{10}$ the concentrations that have been reported to cause alterations in the mobility and topography of cell surface receptors.     

A rapid assay of acyl-coenzyme A:lysolecithin acyltransferase activity

A simple and rapid procedure for the assay of acyl-coenzyme A:1-acyl-sn-glycero-3-phosphocholine acyltransferase (lysolecithin acyltransferase, LLAT [EC 2.3.1.23]) activity in crude enzyme preparations is described. The incubation system utilizes lysolecithin and [1-14C]-oleoyl-coenzyme A as substrates. Labeled fatty acid released due to accompanying acyl-coenzyme A hydrolase [EC 3.1.2.2]activity is first removed by di-isopropyl ether extraction. The labeled lecithin produced due to LLAT action is then quantitatively recovered by partition of the incubation medium with di-isopropyl ether-n-butanol 60:40 (v/v). Selective extraction of the labeled lecithin formed and avoidance of customary thin-layer chromatographic isolation procedures permits assay of LLAT activity with excellent accuracy at a substantial saving of time. The entire assay can be completed in less than 30 min as compared to 2-3 hrs when following conventional procedures.     

Substrate specificity of plasma lysolecithin acyltransferase and the molecular species of lecithin formed by the reaction

Human plasma lecithin-cholesterol acyltransferase also converts lysolecithin to lecithin in the presence of low density lipoproteins. To understand the physiological importance of this lysolecithin acyltransferase reaction, we investigated the molecular species of lysolecithin available for acylation in normal plasma and the lecithins which are formed by the acylation of each of these lysolecithins. Palmitate- and stearate-containing lysolecithins were formed by the lecithin-cholesterol acyltransferase reaction, whereas oleate- and linoleate-containing lysolecithins were formed by the action of post-heparin lipase(s). All the natural lysolecithins were esterified at comparable rates by the isolated enzyme. Lyso platelet-activating factor was esterified about 70% as efficiently as the lysolecithins, while lysophosphatidylethanolamine was esterified at about 30% the rate observed with lysolecithin. The 2-acyl isomers of lysolecithin were acylated to the same extent as the 1-acyl isomers, although considerable isomerization of the former took place during the incubation. There were no net changes in the concentrations of lecithin and lysolecithin after 6 h of incubation with the enzyme, although over 10% of the labeled lysolecithin was converted to lecithin, indicating that the endogenous lecithin serves as the acyl donor in the reaction. When the molecular species of lecithin formed were analyzed by high performance liquid chromatography, the same pattern of fatty acid incorporation was observed with all the lysolecithins used. The bulk of the radioactivity was incorporated into molecular species formed by the acylation with linoleic, oleic, and palmitic acids, in decreasing order. However, in each case, the lecithins formed by acylation with palmitic acid had the highest specific radioactivity, followed by those acylated with linoleic and oleic acids. From these results it is postulated that the enzyme alters the molecular species composition of lecithin in plasma without increasing the net amount of total lecithins.     

Induction of prostanoid synthesis in human platelets by the late complement components C5b-9 and channel forming antibiotic nystatin: inhibition of the reacylation of liberated arachidonic acid

Treatment of human platelets by the purified late complement components C5b-9 results in a dose- and time-dependent release of prostaglandin E (PGE) and thromboxane B2 (TXB2). To study the mechanism underlying the complement-induced prostanoid synthesis, we examined whether C5b-9 affected the enzyme acyl-coA:lysolecithin acyltransferase (E.C.2.3.1.2.3) that catalyzes the reinsertion of liberated arachidonic acid, the precursor molecule of the prostanoids. With C5b-9 doses sufficient to induce prostanoid synthesis, the activity of lysolecithin acyltransferase, measured as conversion of lysophosphatidyl choline to phosphatidyl choline, was inhibited. For comparison, another channel-forming substance, nystatin, was studied. Nystatin had an effect similar to C5b-9: PGE and TXB2 release was stimulated, whereas acyltransferase activity was inhibited. These finding support the concept that inhibition of lysolecithin acyltransferase might be the prerequisite for prostanoid production.     

Phospholipid metabolism of stimulated lymphocytes: Comparison of the activation of acyl-CoA: Lysolecithin acyltransferase with the binding of concanavalin A to thymocytes

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA: $\text{1-acylglycero-3-phosphorylcholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23) was determined as also the binding of ¹²⁵I-labelled concanavalin A to intact cells and isolated membranes.The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 °C.The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Corespondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8).These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.     

Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad.

Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol.     

Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

Quantitative studies on lysolecithin-mediated hemolysis. Use of ether-deoxy lysolecithin analogs with varying aliphatic chain-lengths

The process of lysolecithin-mediated hemolysis has been investigated by use of various ether-deoxy lysolecithin analogs (1-alkyl-propanediol-3-phosphorylcholine) with alkyl residues of 10-22 carbon atoms. Hemolytic activities were defined either as molar amounts to be added for 50% lysis (L50) or as cell-bound amounts at 50% lysis (A50). It was found, that in contrast to L50, A50 values are independent of experimental conditions. Moreover, L50 values primarily reflect the binding affinities, while A50 values give more accurate information on the actual membrane-disturbing potential. The strongest hemolytic C16-lysolecithin analog required 2 - 10(7) or 5 - 10(7) molecules bound per cell for 50% lysis at 0 or 37degrees C, respectively, corresponding to about 10 or 25% of the total membrane phospholipids. Evidence is presented, indicating that (a) lysophosphatides bind to cells below their critical micelle concentration, (b) micelles themselves are not generally necessary for cell lysis. Red cells of different species (man and cattle) as well as at varying temperatures exhibit significantly different sensitivities in terms of L50 and A50 values. These differences, however, depend on the degree of hydrophobicity of the lysolecithins and disappear in the case of lysolipids having C10 or C12 aliphatic residues. The data are in agreement with our hypothesis that cellular sensitivity to lysolecithin lysis may be determined by the degree of segregation of lysolecithin-rich areas within the membrane lipid phase.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.     

Effect of apoprotein B conformation on the activation of lysolecithin acyltransferase and lecithin: cholesterol acyltransferase. Studies with subfractions of low density lipoproteins.

In order to determine the role of apoprotein (apo) B conformation in the activation of the lysolecithin acyl-transferase reaction, we studied the activation of purified enzyme by various subfractions of low density lipoprotein (LDL), isolated by density gradient centrifugation. The activation of LAT correlated positively with the density of LDL and negatively with cholesterol/protein and triglyceride (TG)/protein ratios. The enzyme activation was also positively correlated with the number of trinitrobenzenesulfonic acid-reactive lysine amino groups, which increased with increasing density of LDL. The immunoaffinity of the LDL subfractions for B1B6, a monoclonal antibody directed to the receptor-binding region of apoB, increased with increasing density, while the affinity toward C1.4, a monoclonal antibody directed to the amino-terminal region of apoB, was not altered. Enrichment of normal whole LDL with TG resulted in a 45% reduction in enzyme activation, a 27% decrease in the number of trinitrobenzenesulfonic acid-reactive lysine groups, and a marked reduction in the immunoaffinity for B1B6. All these parameters reversed to normal when the TG-enriched LDL was treated with milk lipoprotein lipase, which specifically reduced the TG content of LDL. The LDL subfractions also supported cholesterol esterification by the purified enzyme, in parallel with lysolecithin esterification, indicating that apoB can also serve as an activator of the lecithin-cholesterol acyltransferase reaction. These results strongly suggest that the localized conformational change of apoB which occurs during the TG depletion of the precursor particle is critical for its activation of acyltransferase reactions, in a manner analogous to its interaction with the cellular receptors.     

Lysolecithin:lysolecithin acyltransferase from rabbit lung. A conformational study

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity. The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9-6.4 was essential for denaturation. The structure was stable at basic pH. The addition of lipids resulted in a non-specific stabilization of the disordered conformation, in the same manner as the addition of NaCl. From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.     

Intramicrosomal location in intestine of enzymes which synthesize complex lipid.

Smooth and rough microsomal fractions were prepared from hamster intestinal mucosa and assayed for RNA, diglyceride acyltransferase, cholinephosphotransferase and lysolecithin acyltransferase. The specific activity of cholinephosphotransferase was 4-fold more in the rough than in the smooth microsomal fractions, whereas diglyceride and lysolecithin acyltransferases were respectively 26 and 57% more active in the rough microsomal fraction. The two acyltransferases were similarly located in the microsomes and more closely corresponded to the location of dietary lipid than cholinephosphotransferase.     

The binding of monoribosomes, oligoribosomes and polyribosomes to reticular membranes from rat liver.

Proteoglycans from pig laryngeal cartilage prepared by dissociative extraction in guanidine hydrochloride were studied in dilute solution by light-scattering and ultracentrifugation. In buffered 150mM-NaCl, pH7.4, the proteoglycan particle weights were about 5x10(6) daltons, but at 100mM-, 200mM- and 300mM-NaCl particle weights of 2.5x10(6)--3.0x10(6) daltons were observed. These results, together with corroborative evidence from sedimentation-velocity experiments, were interpreted in terms of proteoglycans self-associating at physiological ionic strength. The data were examined by using a proteoglycan monomer-dimer model. Proteoglycan preparations that had thiol groups partially carboxymethylated gave particle weights of 3.2x10(6)--3.5x10(6) daltons in 150mM-NaCl, which suggested that carboxymethylation inhibited multimerization and hence that the protein core is implicated in the binding site. Further studies showed that the multimers were stable to 60 degrees C, unlike the hyaluronate-proteoglycan complex.     

Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.

The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.     

Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast.     

Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle

Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed.